Self-renewing intestinal epithelium requires layers of precise control for homeostasis. Amazingly, intestinal epithelium completely renews itself every five days, with multiple cell types maintained at just the right ratios throughout an individual's entire life. However, our understanding of how epithelium maintains such robust organization is far from complete. Lack of this exquisite homeostatic control is the basis for a variety of diseases, including Crohn's disease, irritable bowel syndrome, ulcerative colitis, and gastrointestinal cancers. GSK-3 is a quintessential signaling hub in intestinal tissue homeostasis: it receives and deciphers multiple upstream microenvironmental signals, selectively affects multiple downstream cellular processes, and alters cellular response to drugs. Despite GSK-3's presence in many cellular processes, its function remains unclear and understudied. We lack a systematic understanding of when and where GSK-3 affects the cascade of signals from the microenvironment through molecular networks to cellular decisions in the context of complex epithelial tissues. Here, we propose to study the role of GSK-3 as a hub for signal transduction and maintenance of tissue homeostasis as well as identify drug classes whose effects depend on GSK-3 activities in physiologically relevant conditions. To accomplish this, we have developed: innovative organotypic models of gut epithelium that are ideal for image-based perturbation assays; live-cell reporters of GSK-3 activity; and quantitative, single-cell approaches for deciphering GSK-3's role in transforming microenvironmental signals to homeostatic decisions. Together, we propose to: (Aim 1) identify GSK-3 interaction networks that regulate gut homeostasis; (Aim 2) understand GSK-3's role in signal insulation and crosstalk; and (Aim 3) elucidate how GSK-3 activity affects drug response.